During the past year, we developed new method to detect oxidative stress of outer retina in live animal. In our previous study, we have found that light stimulation causes a significant increase in hydration of outer retina and expansion of distance between external limiting membrane (ELM) and retinal pigment epithelium (RPE). This light-induced outer retina expansion can be measured in vivo non-invasively by optical coherence tomography (OCT). Applied a QUEST strategy with ultrahigh-resolution OCT imaging, we tested the hypothesis that correcting oxidative stress suppressed light-evoked expansion of outer retina with antioxidants (AO) is a novel way to encode oxidative stress information into the OCT image. C57BL/6 mice were maintained in the dark for 20 hours and studied by OCT before and after 1 hour of light exposure. Outer retina (OR, from outer limiting membrane to the retinal pigment epitheliumchoroid boundary) thickness in dark or light were measured, and the light-dark difference (i.e., the photoresponse) was calculated. Subgroups of mice were given either saline or d-cis-diltiazem (an inducer of transient and non-damaging OR oxidative stress) AO's (methylene blue given 24 hours prior to examination and -lipoic acid applied 1 hour prior to examination); one group was given only AOs but kept in the dark. OR thicknesses of controls were 54.2 0.7 um (mean SEM) in dark and 57.7 0.7 um after light-exposure. OR thickness of dark-adapted mice was not affected by AOs (54.8 0.8 um, P > 0.05). In a d-cis-diltiazem-treated mouse, light OR thickness appeared similar to that in the dark, suggesting an impaired OR photoresponse. Light-exposed d-cis-diltiazem injected mice had an OR thickness of 53.0 0.5 um, which was different (P < 0.0001) from control values. In dark-adapted d-cis-diltiazem-treated mice, OR thickness was 53.6 0.5 um, which was not different from the other dark OR thicknesses. d-cis-Diltiazem prevented the light-induced extension of OR thickness. Averaged light-induced OR thickness change was-0.6 0.4 um, which was not different (P = 0.14) from 0 and was smaller (P < 0.0001) than those observed in control animals (3.5 0.5 um). Treatment with AOs restored the photoresponse. In light-exposed mice given both d-cis-diltiazem and AOs, the OR thickness was 55.9 0.7 um, which is not different (P = 0.4) from controls. In dark-adapted d-cis-diltiazem and AO-treated mice, OR thickness was 52.6 0.7 um, which was not different from the other dark OR thicknesses. d-cis-Diltiazem mice treated with AOs showed a light-evoked OR expansion of 3.3 0.6 um, which is different (P < 0.0001) from 0 but not from controls (P = 1.0). In summary, we demonstrated for the first time of a new functionality for OCT: the ability to measure localized oxidative stress in vivo. This study carefully built on our previous findings that light-evoked expansion of the OR is an essential physiology measurable by OCT, and that light-evoked expansion of the OR is sensitive to oxidative stress. The presents results are important because they address a longstanding problem, namely, that conventional assays are unable to noninvasively measure OR oxidative stress in vivo and potentially in patients to personalize antioxidant treatment options. We also reported that combining QUEST OCT with an acute administration of d-cis-diltiazem is a unique way to noninvasively evaluate OR AO defenses. Finally, we found that the OR thickness in the light alone can be used to detect OR oxidative stress, an observation that may help facilitate translation of QUEST OCT into a clinical setting. QUEST OCT appears to be a promising new clinically relevant tool for early diagnosis and individualized AO treatment in sight-threatening diseases with an OR oxidative stress etiology.